We have developed a new microspectrophotometric method for blood O2 saturation determination in the small blood vessels of quick-frozen brain. Combining this information of arterio-venous O2 saturation difference in the micro-regions of brain, with regional blood flow determined by the 14C-iodoantipyrene method we shall determine O2 consumption in the various micro-regions of brain by the application of Fick Principle. There are no other methods for determining O2 consumption in the micro-regions of brain. The need and usefulness of being able to monitor the O2 metabolic rates of various parts of brain in relation to mental activity is well recognized. This new method will enable us to determine variations in O2 consumption rates in the various micro-regions of brain of anesthetized animals, awake animals, animals in non-rapid-eye-movement sleep and animals in rapid-eye-movement sleep. From the alterations in regional cerebral metabolic rate we shall be able to determine which brain structures are involved in sleep-wakefulness process.